GABAC receptor binding of quantum-dot conjugates of variable ligand valency

Bioconjug Chem. 2010 Aug 18;21(8):1455-64. doi: 10.1021/bc100050s.

Abstract

Highly fluorescent CdSe quantum dots (qdots) can serve as a platform for tethering multiple copies of a receptor-targeted ligand, affording study of how the level of multivalency affects receptor binding. We previously showed that qdots conjugated with long PEG chains terminated by muscimol, a known GABA(C) agonist, exhibit specific binding to the surface membrane of GABA(C) receptor-expressing Xenopus oocytes. The present report addresses the effect of varying the number, i.e., valency, of muscimol- (M-) terminated PEG chains attached to the qdot on binding of the resulting conjugate to GABA(C) receptors. M-PEG-qdots of differing muscimol valency were prepared by conjugating AMP-CdSe/ZnS qdots with muscimol-terminated and methylamine-terminated PEG chains in proportions designed to yield varying percentages of muscimol-terminated chains among the total approximately 150-200 chains bound to the qdot. The investigated valencies represented 0%, approximately 25%, approximately 50%, and 100% loading with muscimol (preparations termed M-PEG-qdot0, M-PEG-qdot25, M-PEG-qdot50, and M-PEG-qdot100, respectively. Binding of a given conjugate to surface membranes of GABA(C) receptor-expressing oocytes was analyzed by quantitative fluorescence microscopy following defined incubation with approximately 30 nM of the conjugate. With 5-20 min incubation, the fluorescence signal resulting from incubation with M-PEG-qdot25 exceeded, by approximately 6-fold, the fluorescence level obtained with M-PEG-qdot preparations that lacked muscimol-terminated chains (M-PEG-qdot0). M-PEG-qdot50 yielded a net signal roughly similar to that of M-PEG-qdot25, and that produced by M-PEG-qdot100 exceeded, by approximately 30-50%, those for M-PEG-qdot25 and M-PEG-qdot50. The time course of changes in oocyte surface membrane fluorescence resulting from the introduction of and removal of M-PEG-qdots in the medium bathing the oocyte indicated only a modest dependence of both binding and wash-out kinetics on muscimol valency. The results demonstrate a dependence of the binding activity of the M-PEG-qdot conjugates on muscimol valency, presumably reflecting higher GABA(C) avidity and/or affinity of the muscimol at high valency, and provide insight on the interactions of membrane receptor proteins with qdot conjugates containing multiple copies of a receptor-targeting ligand.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cadmium Compounds / chemistry*
  • Fluorescence
  • Ligands
  • Methylamines / chemistry
  • Molecular Structure
  • Muscimol / chemistry
  • Polyethylene Glycols / chemistry
  • Quantum Dots*
  • Receptors, GABA / chemistry*
  • Selenium Compounds / chemistry*
  • Sulfides / chemistry
  • Zinc Compounds / chemistry

Substances

  • Cadmium Compounds
  • GABA-C receptor
  • Ligands
  • Methylamines
  • Receptors, GABA
  • Selenium Compounds
  • Sulfides
  • Zinc Compounds
  • Muscimol
  • Polyethylene Glycols
  • cadmium selenide
  • methylamine
  • zinc sulfide